Many behavioral, cellular and biochemical processes are controlled by an endogenous circadian clock, and the molecular mechanisms underlying these self-sustaining circadian clocks are of interest to both clinicians and basic scientists. The clinical relevance of circadian rhythms is most obvious in their control of sleep/wake cycles, but many other medically relevant processes are also regulated by circadian rhythms. Endocrine function, cancer regulation, psychiatric disorders and drug metabolism are all affected by circadian clocks, and therefore knowledge of their basic mechanism can contribute to many areas of modern medicine. At the core of this molecular mechanism in mammals and flies are oscillations of the PER protein, which are controlled at the post-translational level by the activities of several kinases, including the DBT casein kinase I. This application proposes to elucidate the mechanism by which DBT regulates the oscillations of PER. The first aim will identify other proteins which interact with DBT and PER to regulate the activity of DBT and bring about the biochemical consequences of PER's phosphorylation by DBT. This aim will employ both a Drosophila cell culture line (S2 cells) as well as adult fly heads. Aim 2 will identify the sites within PER that are phosphorylated by DBT and other kinases, under various conditions relevant to circadian function in the adult fly head. A mass-spectrometry-based proteomic approach will be a major focus of both Aims 1 and 2. Finally, Aim 3 will address how protein/protein interactions and phosphorylation at particular sites with PER regulate processes involving PER and DBT. Aim 3 will therefore address the biological consequences of the interactions and phosphorylation sites identified in Aims 1 and 2. PUBLIC HEALTH RELEVANCE: Circadian rhythms are daily cycles of behavior and physiology, as exemplified by our sleep/wake behavior, and they affect drug efficacy, cancer, metabolism and psychological disorders. This application seeks to understand one of the key biochemical mechanisms that produce the circadian clock. The mechanism is very amenable to treatment with drugs, so the understanding may affect treatments of a number of human diseases.